Bottom Line:
We also show that IMC1b-deficient ookinetes display abnormal cell shape, reduced gliding motility, decreased mechanical strength, and reduced infectivity.The similarities observed between the loss-of-function phenotypes of IMC1a and IMC1b show that membrane skeletons of ookinetes and sporozoites function in an overall similar way.However, the fact that ookinetes and sporozoites do not use the same IMC1 protein implies that different mechanical properties are required of their respective membrane skeletons, likely reflecting the distinct environments in which these life stages must operate.

Affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom.

ABSTRACTMembrane skeletons are cytoskeletal elements that have important roles in cell development, shape, and structural integrity. Malaria parasites encode a conserved family of putative membrane skeleton proteins related to articulins. One member, IMC1a, is expressed in sporozoites and localizes to the pellicle, a unique membrane complex believed to form a scaffold onto which the ligands and glideosome are arranged to mediate parasite motility and invasion. IMC1b is a closely related structural paralogue of IMC1a, fostering speculation that it could be functionally homologous but in a different invasive life stage. Here we have generated genetically modified parasites that express IMC1b tagged with green fluorescent protein, and we show that it is targeted exclusively to the pellicle of ookinetes. We also show that IMC1b-deficient ookinetes display abnormal cell shape, reduced gliding motility, decreased mechanical strength, and reduced infectivity. These findings are consistent with a membrane skeletal role of IMC1b and provide strong experimental support for the view that membrane skeletons form an integral part of the pellicle of apicomplexan zoites and function to provide rigidity to the pellicular membrane complex. The similarities observed between the loss-of-function phenotypes of IMC1a and IMC1b show that membrane skeletons of ookinetes and sporozoites function in an overall similar way. However, the fact that ookinetes and sporozoites do not use the same IMC1 protein implies that different mechanical properties are required of their respective membrane skeletons, likely reflecting the distinct environments in which these life stages must operate.

Mentions:
Upon closer examination, the morphology of IMC1b-KO ookinetes appeared abnormal (Fig. 4A). Compared with WT ookinetes, IMC1b-KO ookinetes were typically shorter (mean length 10.76 ± 0.15 μm for WT; 9.27 ± 0.10 μm for IMC1b-KO; n = 100), and wider (mean width 1.98 ± 0.03 μm for WT; 2.67 ± 0.05 μm for IMC1b-KO; n = 100). In particular, IMC1b-KO ookinetes possessed a bulging area typically in the central part of the cell (Fig. 4A). As expected, GFP was expressed in these ookinetes but was no longer targeted to the IMC/SPN resulting in cytoplasmic green fluorescence (Fig. 4A). To assess infectivity of IMC1b-KO parasites, mosquitoes were infected and analyzed for oocyst development. Oocyst numbers in IMC1b-KO parasite-infected mosquitoes were 8–10-fold lower than in WT parasite-infected mosquitoes (Table 1). These oocysts did, however, appear morphologically normal and produced sporozoites that successfully invaded the salivary glands. These sporozoites were infectious to mice following mosquito bite (data not shown). This is consistent with the observation that IMC1b is not expressed downstream of the ookinete stage (Fig. 2). Thus, parasites are capable of completing the Plasmodium life cycle without functional IMC1b, albeit at reduced efficiency. IMC1b-KO parasites that had been passaged through mosquitoes retained their loss-of-function phenotype both in terms of cell shape (data not shown) and infectivity (Table 1).

Mentions:
Upon closer examination, the morphology of IMC1b-KO ookinetes appeared abnormal (Fig. 4A). Compared with WT ookinetes, IMC1b-KO ookinetes were typically shorter (mean length 10.76 ± 0.15 μm for WT; 9.27 ± 0.10 μm for IMC1b-KO; n = 100), and wider (mean width 1.98 ± 0.03 μm for WT; 2.67 ± 0.05 μm for IMC1b-KO; n = 100). In particular, IMC1b-KO ookinetes possessed a bulging area typically in the central part of the cell (Fig. 4A). As expected, GFP was expressed in these ookinetes but was no longer targeted to the IMC/SPN resulting in cytoplasmic green fluorescence (Fig. 4A). To assess infectivity of IMC1b-KO parasites, mosquitoes were infected and analyzed for oocyst development. Oocyst numbers in IMC1b-KO parasite-infected mosquitoes were 8–10-fold lower than in WT parasite-infected mosquitoes (Table 1). These oocysts did, however, appear morphologically normal and produced sporozoites that successfully invaded the salivary glands. These sporozoites were infectious to mice following mosquito bite (data not shown). This is consistent with the observation that IMC1b is not expressed downstream of the ookinete stage (Fig. 2). Thus, parasites are capable of completing the Plasmodium life cycle without functional IMC1b, albeit at reduced efficiency. IMC1b-KO parasites that had been passaged through mosquitoes retained their loss-of-function phenotype both in terms of cell shape (data not shown) and infectivity (Table 1).

Bottom Line:
We also show that IMC1b-deficient ookinetes display abnormal cell shape, reduced gliding motility, decreased mechanical strength, and reduced infectivity.The similarities observed between the loss-of-function phenotypes of IMC1a and IMC1b show that membrane skeletons of ookinetes and sporozoites function in an overall similar way.However, the fact that ookinetes and sporozoites do not use the same IMC1 protein implies that different mechanical properties are required of their respective membrane skeletons, likely reflecting the distinct environments in which these life stages must operate.

Affiliation:
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom.

ABSTRACTMembrane skeletons are cytoskeletal elements that have important roles in cell development, shape, and structural integrity. Malaria parasites encode a conserved family of putative membrane skeleton proteins related to articulins. One member, IMC1a, is expressed in sporozoites and localizes to the pellicle, a unique membrane complex believed to form a scaffold onto which the ligands and glideosome are arranged to mediate parasite motility and invasion. IMC1b is a closely related structural paralogue of IMC1a, fostering speculation that it could be functionally homologous but in a different invasive life stage. Here we have generated genetically modified parasites that express IMC1b tagged with green fluorescent protein, and we show that it is targeted exclusively to the pellicle of ookinetes. We also show that IMC1b-deficient ookinetes display abnormal cell shape, reduced gliding motility, decreased mechanical strength, and reduced infectivity. These findings are consistent with a membrane skeletal role of IMC1b and provide strong experimental support for the view that membrane skeletons form an integral part of the pellicle of apicomplexan zoites and function to provide rigidity to the pellicular membrane complex. The similarities observed between the loss-of-function phenotypes of IMC1a and IMC1b show that membrane skeletons of ookinetes and sporozoites function in an overall similar way. However, the fact that ookinetes and sporozoites do not use the same IMC1 protein implies that different mechanical properties are required of their respective membrane skeletons, likely reflecting the distinct environments in which these life stages must operate.